The invention is related to the area of mismatch repair genes. In particular it is related to the field of mutagenesis.
Within the past four years, the genetic cause of the Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynch syndrome II, has been ascertained for the majority of kindreds affected with the disease (13). The molecular basis of HNPCC involves genetic instability resulting from defective mismatch repair (MMR). Many genes have been identified in rodents and humans that encode for proteins that appear to participate in the MMR process, including the mutS homologs GTBP, hMSH2, and hMSH3 and the mutL homologs hMLH1, hPMS1, and hPMS2 (2, 7, 11, 17, 20, 21, 22, 24). Germ line mutations in four of these genes (hMSH2, hMLH1, hPMS1, and hPMS2) have been identified in HNPCC kindreds (2, 11, 13, 17, 24). Though the mutator defect that arises from the MMR deficiency can affect any DNA sequence, microsattelite sequences are particularly sensitive to MMR abnormalities (14). Microsattelite instability is therefore a useful indicator of defective MMR. In addition to its occurrence in virtually all tumors arising in HNPCC patients, Microsattelite instability is found in a small fraction of sporadic tumors with distinctive molecular and phenotypic properties (27).
HNPCC is inherited in an autosomal dominant fashion, so that the normal cells of affected family members contain one mutant allele of the relevant MMR gene (inherited from an affected parent) and one wildtype allele (inherited from the unaffected parent). During the early stages of tumor development, however, the wildtype allele is inactivated through a somatic mutation, leaving the cell with no functional MMR gene and resulting in a profound defect in MMR activity. Because a somatic mutation in addition to a germline mutation is required to generate defective MMR in the tumor cells, this mechanism is generally referred to as one involving two hits, analogous to the biallelic inactivation of tumor suppressor genes that initiate other hereditary cancers (11, 13, 25). In line with this two hit mechanism, the non-neoplastic cells of HNPCC patients generally retain near normal levels of MMR activity due to the presence of the wildtype allele.
A wide range of organisms with defective MMR have been found to have widespread genetic mutations throughout their genome. In all cases, these organisms have germline mutations within both copies of a particular MMR gene. Recently, work done by Nicolaides et al have shown that a decrease in MMR can be achieved within cells from higher order organisms by introducing a dominant negative allele of a MMR gene. These data suggest that the use of such an approach can generate genetically altered organisms to produce new output traits. There is a need in the art for additional methods with which to generate genetic diversity.
It is an object of the present invention to provide a method for rendering cells hypermutable.
It is another object of the present invention to provide genetically altered cell lines.
It is another object of the present invention to provide phenotypically altered cell lines.
It is yet another object of the present invention to provide a method to produce an enhanced rate of genetic hypermutation in a cell.
It is a further object of the invention to provide a method of mutating a gene(s) of interest in a cell.
It is a further object of the invention to claim composition of matter for a genetically altered bacterial purine phosphorlyase.
It is a further object of the invention to claim composition of matter for a genetically altered bacterial purine phosphorlyase as a diagnostic tool for monitoring mismatch repair deficiency of a eucaryotic cell.
It is a further object of the invention to claim composition of matter for a generating genetically altered genes by incorporating a polymononucleotide tract to measure for altered mismatch repair in eucaryotic cells.
Yet another object of the invention is to provide a method of creating cells with new phenotypes.
Yet another object of the invention is to provide a method of creating cells with new phenotypes and a stable genome.
Yet another object of the invention is to provide a method of regulating the genetic stability of a cell or organism""s genome.
It is a further object of the invention to generate hypermutable cell lines using inducible vectors containing dominant negative mismatch repair gene mutants.
It is a further object of the invention to screen for hypermutable cell lines containing inducible vectors with dominant negative mismatch repair gene mutants under induced gene expression conditions.
It is a further object of the invention to screen for hypermutable cell lines containing inducible vectors with dominant negative mismatch repair gene mutants under induced gene expression conditions for altered gene structure and/or new phenotypes.
It is a further object of the invention to turn off expression of a dominant negative MMR gene in cells containing structurally altered target genes and/or new phenotypes to restore genomic stability.
It is a further object of the invention to screen hypermutable cell lines containing an inducible vector comprising a dominant negative mismatch repair gene mutant under inducing conditions in the presence of chemical mutagens or ionizing radiation for structurally altered target genes and/or new phenotypes. Cells containing altered gene structure and/or new phenotype are then removed from inducer molecule and genetic stability is restored.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention, a method for making a hypermutable cell is provided. A polynucleotide encoding a dominant negative allele of a mismatch repair gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction of the gene.
In another embodiment of the invention, an isolated hypermutable cell will be provided. The cell comprises a dominant negative allele of a mismatch repair gene. The cell is exposed to DNA akylating agents. The cell exhibits an enhanced rate of hypermutation.
In another embodiment of the invention, a method is provided for introducing a mutation into a gene of interest. A polynucleotide encoding a dominant negative allele of a mismatch repair gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction of the gene. The cell further comprises a gene of interest. The cell is grown. The cell is tested to determine whether the gene of interest harbors a mutation.
In another embodiment of the invention, a method is provided for inserting a polymononucleotide tract in a gene to measure for mismatch repair activity of a eucaryotic cell. A polynucleotide tract is inserted out-of-frame into the coding region of a gene or a cDNA. The gene is introduced into a cell. The polymononucleotide tract is altered by mismatch repair deficiency. An in-frame altered gene is produced.
In another embodiment of the invention, a method is provided for producing new phenotypes of a cell. A polynucleotide encoding a dominant negative allele of a mismatch repair gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction of the gene. The cell is grown. The cell is tested for the expression of new phenotypes. Another embodiment of the invention is the use of cells containing an inducible vector consisting of a dominant negative mismatch repair gene mutants under inducing conditions in the presence of chemical mutagens or ionizing radiation for altered target genes and/or new phenotypes. Cells containing altered gene structure and/or new phenotype are then removed from inducer molecule and genetic stability is restored. The cells are now used for commercial properties such as but not limited to recombinant manufacturing and/or gene discovery.
Another embodiment of the invention is the use of MMR defective cells containing a gene of interest in the presence of chemical mutagens or ionizing radiation for altered target genes and/or new phenotypes. Cells containing altered gene structure and/or new phenotype are then stably transduced with a wildtype MMR complementing gene and genetic stability is restored. The cells are now used for recombinant manufacturing or gene discovery.
In another embodiment of the invention, a method is provided for restoring genetic stability in a cell with defective mismatch repair gene. The activity of the mismatch repair process is restored and its genome is stable.
In another embodiment of the invention, a method is provided for restoring genetic stability in a cell with defective mismatch repair activity and a newly selected phenotype. The MMR deficiency can occur through the inactivation of endogenous MMR genes via genomic mutations or through the introduction of an eucaryotic expression vector producing a dominant negative MMR gene allele. In the case of cells lacking endogenous MMR due to a defect in an endogenous MMR gene, the cell is selected for a new phenotype or altered gene, RNA, or polypeptide. The cell becomes genetically stable through the introduction of a normal functioning MMR gene that complements the genomic defect of the host cell. This complementation group can include the use of any gene known to participate in mismatch repair deficiency. In the case were the expression of the dominant negative mismatch repair gene is used to induce DNA hypermutability, the dominant negative MMR gene expression will be suppressed by removal of the inducer molecule or by knocking out the expression of the dominant negative gene allele using standard gene knockout technology used by those skilled in the art (Waldman, T., et.al. Cancer Res 55:5187-5190, 1995). In any case, the cell restores its genetic stability and the new phenotype is stable.
These and other embodiments of the invention provide the art with methods that can generate enhanced mutability in organisms, cells and animals as well as providing genetically altered stable organisms cells and animals harboring potentially useful genome alterations.
The use of a dominant negative MMR gene allele is important in generating global mutations throughout the genome of a host organism in a regulated fashion. While the use of dominant negative alleles have been previously demonstrated to be capable of inducing global mutagenesis in a wide range of hosts (bacteria, yeast, mammals, plants) the use of inducible vectors to turn the dominant negative MMR gene mutant on to generate genome-wide mutation followed by selection for new biochemical output traits (e.g., resistance to chemical mutagens) and turning off of the dominant negative MMR gene allele to restore genetic stability is a new aspect of the invention. This method is now suitable for generating genetically diverse prokaryotic, eucaryotic and mammalian cells that can be screened for genetic mutations in genes involved in new phenotypes. In addition, this application teaches of the use of introducing dominant negative MMR alleles under control of inducible expression elements into MMR proficient cells. Stable or transiently transduced cells are then exposed to inducer molecule resulting in expression of the dominant negative MMR gene. Expression of the dominant negative product interferes with the endogenous MMR machinery, thereby causing genetic instability that leads to genetically diverse sublines. These cells are then put under specific selective assays and screened for new phenotypes and/or altered gene structures. After the establishment of sublines containing altered target genes and/or new phenotypes, cells are then rendered genetically stable by removal of the inducer molecule and a stable cell line is now produced that contains an altered gene and/or exhibits a new phenotype. This cell line can be used for gene discovery, drug target discovery, recombinant gene mutagenesis, and/or recombinant protein production.
It is well established that MMR deficient organisms are more tolerant to DNA damaging agents such as alkylating agents or ionizing radiation thereby leading to enhanced levels of genome-wide or locus-specific mutagenesis. Here we teach the use of exposing cell lines expressing dominant negative MMR under control of an inducible expression element to DNA damaging agents that can lead to enhanced genome wide mutagenesis. Cell lines are then screened for mutations in target genes or screened for novel phenotypes. Sublines with altered genes or phenotypes are then removed from inducer agent to xe2x80x9cturn offxe2x80x9d the dominant negative MMR gene allele to restore genetic stability. This cell line can be used for gene discovery, drug target discovery, recombinant gene mutagenesis, and/or recombinant protein production.
Finally, the use of mammalian cell lines that are naturally defective for MMR can be used to introduce a plasmid containing a gene of interest. The gene can be introduced and expressed transiently or stably. The cell now grows and the structure and/or function of the introduced gene is screened to identify those with structural and/or functional alterations. To enhance mutation rate, cells can be further exposed to DNA damaging agents such as but not limited to alkylating chemical mutagens or ionizing radiation to produce enhanced genome wide mutation rate in the host. Once a cell line(s) containing mutations within the gene of interest are generated, the cell is stably transduced with a gene that complements for the endogenous MMR defect. The cell line is now genetically stable and the cell line is suitable for producing altered gene products for gene discovery, recombinant gene mutagenesis, and/or recombinant protein production.